The investigation of enzyme-catalyzed proton transfer reactions of thiol esters is proposed. The enzyme thiolase catalyzes the condensation of two molecules of acetyl-Coenzyme A (a thiol ester) to form Coenzyme A and acetoacetyl-Coenzyme A. The mechanism involves condensation of an acetyl-enzyme with a carbanion or carbanion-like species derived by abstraction of a proton alpha- to the carbonyl group of a thiol ester. The exchange of hydrogen isotopes (both deuterium and tritium) into and out of substrate and product will be studied under initial velocity conditions where the acetoacetyl Coenzyme A product is trapped by enzymatic reduction. Chemical modification of the active site sulfhydryl group will be used to block acyl transfer and condensation reactions so that the enzyme can catalyze only proton exchange. The various exchange rates of native and modified enzymes will help define the importance of proton abstraction, condensation, and proton equilibration with solvent steps to the native enzyme reaction. The possibility of buffer catalysis of the enzyme-catalyzed exchange reaction is also explored.